Vaccination is one of the most cost-effective means to prevent and to control infectious diseases in both human and animal health. Vaccines contain either modified live antigens such as bacteria, viruses, parasites, recombinant (micro-) organisms or inactivated (non-replicating) antigens such as inactivated viruses, inactivated bacteria, inactivated parasites, subunits of infectious (micro-) organisms, proteins, polysaccharides, peptides, glycoproteins, polysaccharide-protein conjugates, peptide-protein-conjugates, etc. In many cases, the immune response against antigens is too low. In such cases, an adjuvant can be added to stimulate the immune response, and thereby increase the protective action of vaccines.
In the control of existing, emerging and re-emerging infectious diseases, adjuvants play a decisive role. Adjuvants may have different functions such as to increase (protective) immune response, to direct the type of response, to increase the duration of (protective) immunity, to reduce the lag-time to (protective) immunity, to reduce the dose of antigen required and reduce the number of vaccinations needed. By combining an antigen with an adjuvant, levels of immunity may be reached that can not be reached by treatment with antigen alone.
Also, an adjuvant may reduce significantly the dose of antigen needed to reach predefined levels of protection, thereby reducing the costs of goods, transport and storage and increasing production capacity and availability. The inexpensive adjuvant replaces a large portion of the expensive antigen and promotes access to these medicinal products to the poor.
Alum (aluminium hydroxide) is the classical example of a safe adjuvant and is applied in several human vaccines. However, in many cases the adjuvant activity is insufficient and a stronger but at least as safe adjuvant is needed.
Furthermore, sulphate-substituted carbohydrate fatty acid ester derivatives are known. These are commonly called sulpholipid-carbohydrates (SL-carbohydrates), and their synthesis and function as adjuvant are known.
The known SL-carbohydrates have in common that they are prepared by two synthesis steps, an esterification of the hydroxyl-groups of the carbohydrate with the fatty acid(s) and an esterification of the hydroxyl-groups of the carbohydrate with a sulphonating agent. These reactions may be performed simultaneously or in any order. Both reactions are ad random reactions, which means that there is no or limited discrimination between the different hydroxyl groups of the carbohydrate available for esterification. Therefore, the sulphate group(s) and the fatty acid(s) are distributed randomly over the substituted carbohydrate, resulting in a mixture of many different chemical compounds.
The number of chemically distinct SL-carbohydrates formed by ad random synthesis methods depends on the number of reactive sites (hydroxyl groups) on the carbohydrate backbone. Contacting a carbohydrate with 1 equivalent (one mole per mole of carbohydrate) of sulphonating agent results in the formation of carbohydrates with a different number of sulphate groups and in the formation of carbohydrates with sulphate groups at the different positions. For example, SL-carbohydrate mixtures known in the art consist of a mixture of carbohydrate esters, among others with no sulphate group (herein referred to as ‘ZERO’), one sulphate group (herein referred to as ‘MONO’) or multiple sulphate groups (two or more sulphate groups; herein referred to as ‘POLY’). The ratio of ZERO:MONO:POLY can be determined statistically by using the formula disclosed in WO 96/20222. At 1 mole of sulphonating agent per mole of disaccharide (with eight hydroxyl groups available for sulphonation), the theoretical molar ratio of ZERO:MONO:POLY in the final product is 37%:37%:26%. At lower molar ratio of sulphonating agent per carbohydrate, the % POLY formed decreases, and the % ZERO formed increases. At 0.2 mole of sulphonating agent per mole of carbohydrate, the theoretical molar ratio of ZERO:MONO:POLY is 82%:16%:2%.
The use of a mixture of components as a medicinal product may be associated with important drawbacks with respect to quality, consistency and safety of the product. In particular for adjuvants in e.g. vaccines, it is important to emphasize that they are applied at a large scale in healthy subjects. Toxicity, efficacy and quality are therefore important prerequisites.
It is generally accepted that there is a relationship between adjuvant activity (efficacy) and side effects (toxicity) and that increasing efficacy is accompanied by increasing toxicity (see e.g. Hilgers, Methods Mol. Biol. 626 pp. 251-9, 2010). The ratio between efficacy and toxicity is called the E/T ratio, and is an important parameter in defining adjuvants. A drawback of adjuvants comprising a SL-carbohydrate of the prior art is the lack of sufficient safety (too high toxicity or a too low E/T-ratio).
Adjuvant activity and toxicity increase with increasing dose of the adjuvant. By decreasing the dose of adjuvant, the side effects decrease also. However, it is uncertain whether at a level of acceptable toxicity, sufficient efficacy remains. Therefore, there is still a need for adjuvants with a higher efficacy/toxicity-ratio than that of the existing ones, including the SL-carbohydrate-based adjuvants. These improvements may include higher efficacy with similar toxicity, lower toxicity with similar efficacy or higher efficacy combined with lower toxicity.
WO 01/402490 discloses SL-monosaccharides and SL-disaccharides, a method for preparing these SL-monosaccharides and SL-disaccharides and their use as vaccine adjuvant. SL-disaccharide mixtures are formed in a reaction with one molar equivalent of sulphonating agent per mole of disaccharide and seven molar equivalents of fatty acid chlorides (also referred to as acyl chlorides or acoylchloride) per mole of disaccharide. This results in a mixture of 6,561 different possible chemical compounds, the relative presence of which can be determined by statistics. These products have been subject of intense research under the name ‘CoVaccine HT’ (BTG plc, UK). Mixtures obtained by using one or more molar equivalents of sulphonating agent and one or more molar equivalents of fatty acid chloride were tested for their adjuvant activity. However, the toxicity of the tested SL-carbohydrate derivatives was too high for the wide-spread use in humans.
Hilgers et al. (Vaccine 17, pp. 219-228, 1999), Blom & Hilgers (Vaccine 29, pp. 3791-3801, 2011) and EP2580226 disclose undesired side effects such as body temperature rise and local irritation of SL-carbohydrate-based adjuvants and/or vaccines.
Hilgers et al. (Vaccine 17, pp. 222-225, 1999) discloses a relationship between the molecular weight of the SL-carbohydrates on the one hand and adjuvant activity and local reactogenicity at the other. The local reaction but not the adjuvant activity increases with increasing molecular weight of the SL-carbohydrate. Also, a transient increase in body temperature of up to 1.7° C. and swelling at the injection site for up to 4 weeks is very common. Vomiting occurs in 1-25% of cases during the first hour after vaccination. Persistent mild to moderate granulomatous inflammation of the muscular fibers is observed at the injection side up to 8 weeks after vaccination (European Public Assessment Report (EPAR) on porcine circovaccine adjuvanted with SL-cyclodextrin).
EP2580226 discloses adverse events of SL-trisaccharides including local adverse reaction and rise in body temperature. The side effects are significant and prohibit widespread prophylactic application of the product in healthy subjects. Also for a SL-disaccharide, significant local and systemic side-effects were observed also as disclosed by Turkstra et al. (Vaccine 29, pp. 3791-3801, 2011) and EP2580226.
Human clinical trials using SL-disaccharide (CoVaccine HT) have been discontinued for reasons which can be related to or the consequence of the adverse reactions noted with SL-carbohydrates in general, including SL-disaccharides. The known SL-carbohydrate-based adjuvants are considered unsuitable for human use.
WO 2008/005824 discloses compositions comprising natural killer T cell agonists and physiological acceptable vehicles, and methods of stimulating an natural killer T cell and enhancing an immune response. The compounds disclosed are synthetic derivatives of monosaccharide and disaccharide containing a ceramide group and optionally one or multiple sulphate groups. The compounds do not comprise a fatty acid on the saccharide core, and are therefore distinct from the presently claimed compounds.
WO 01/402490 discloses methods to isolate a mixture of SL-monosaccharide and SL-disaccharide derivatives from the reaction mixture. The techniques listed include crystallization, precipitation, filtration, evaporation dialysis or ultrafiltration. Although these methods may be suitable to isolate a mixture of SL-monosaccharide and SL-disaccharide derivatives from the reaction mixture or to separate a mixture of SL-monosaccharide and SL-disaccharide derivatives from certain by-products, they are not suitable to purify MONO isomers of the SL-carbohydrates of the present invention from POLY isomers.
In summary, a drawback of strong adjuvants such as SL-carbohydrate-based products is their relatively high toxicity and a drawback of safe adjuvants such as alum is their relatively low adjuvant activity.
As such, there remains a need for adjuvants with a higher efficacy at the same or lower toxicity, to allow for further increasing the immune response during vaccination. The present invention discloses compounds which achieve that.